HK1075654A1 - Hyperforin derivatives, the use thereof and formulations containing them - Google Patents

Abstract

The use of the reduction products of hyperforin and adhyperforin, pharmaceutically acceptable salts or esters thereof, in the pharmaceutical and/or nutritional field, in particular in the treatment of depression and Alzheimer's disease.

Description

Hyperforin derivatives, their use and formulations containing them

Technical Field

The present invention relates to hyperforin and adhyperforin derivatives and their use in the pharmaceutical and/or nutritional field, in particular in the treatment of depression and alzheimer's disease.

Background

St.John's wort (flowering tops) contains many classes of structurally diverse substances that act directly or indirectly on the central nervous system. The mechanisms of action of these compounds vary, including anti-MAO action (Suzuki OR. et al, Planta Med., 272-4, 1984), action on 5-hydroxytryptamine release and reuptake (Muller W.E. et al, Pharmacophygienry, 30, 102-107, 1997), and benzodiazepine  -like activity (Coot J.M. Pharmacophygienry 30, 108-112, 1997).

Hyperforin, a floroglucin derivative, is one of the main components of the lipophilic fraction of hyperforin perforatum branches; the fraction also contains adhyperforin, which is a higher homologue of hyperforin, although in relatively low concentrations (Erdelmeier c.a.j., pharmacopsory, 31, 2-6, 1998).

Hyperforin: r is CH₃

Adding hyperforin: r is CH₂CH₃

Hyperforin has recently been the subject of many studies that have established its important role as an antidepressant (pharmacoprochytry, 31 suppl.1, 1-60.1998). Furthermore, it is well recognized that extracts of Hypericum perforatum can be used for the prevention and treatment of neurodegenerative diseases, especially Alzheimer's disease (WO/9940905, WO 0057707). In particular, the inorganic cation salts or ammonium salts of hyperforin and adhyperforin are described for this purpose (WO 9941220).

As known from the literature, hyperforin has poor stability under conventional extraction and storage conditions; as described in WO97/13489, the hyperforin content of the aqueous-alcoholic extract of St.John's wort decreased after several weeks. WO97/13489 further describes that in order to obtain a hyperforin-stable extract, an antioxidant must be present throughout the process (extraction, purification and storage). It is therefore evident that the high instability of hyperforin makes the preparation of pharmaceutical formulations of hyperforin very difficult. To overcome this drawback, more stable compounds than hyperforin have recently been prepared, such as the salts and hydroxy-functionalized derivatives disclosed in WO99/41220 (WO 99/64388).

Furthermore, it is known (Bystrov et al, Bioorg. Khim, 1978) that hyperforin and adhyperforin can be converted into the corresponding octahydro derivatives, namely octahydrohyperforin (Ia) and octahydroadhyperforin (Ib),

(Ia：R＝CH₃

Ib：R＝CH₂CH₃)

or by reduction of the keto groups in the 1 and 10 positions to hydroxyl groups with metal hydrides to the corresponding tetrahydro derivatives, namely tetrahydrohyperforin (Ic) and tetrahydrohyperforin (Id).

(Ic：R＝CH₃

Id：R＝CH₂CH₃)

Detailed Description

It has now been found that hyperforin and adhyperforin derivatives obtained by reduction of all the double bonds of the prenyl chain and/or by reduction of the keto groups in the 1 and 10 positions to hydroxyl groups not only have a higher stability, but also have an unexpectedly higher antidepressant, anxiolytic and anti-neurodegenerative activity than hyperforin and adhyperforin.

The object of the present invention is therefore the use of hyperforin and adhyperforin derivatives of formula (I) for the preparation of a medicament, in particular for the preparation of a medicament for the treatment of depression and Alzheimer's disease,

wherein R is methyl or ethyl, R₂Is hydrogen, a pharmaceutically acceptable cation of an inorganic or organic base or a straight or branched chain C₂-C₅An acyl residue, and wherein:

a)R₁is 3-methyl-but-1-yl and oxo groups are present in positions 1 and 10; or

b)R₁Is 3-methyl-2-buten-1-yl and hydroxyl groups are present at the 1 and 10 positions; or

c)R₁Is 3-methyl-but-1-yl and hydroxyl groups are present in the 1 and 10 positions.

a) Preferred compounds of formula (I) as defined in (1) are those wherein R is₂Compounds which are hydrogen, namely octahydrohyperforin (Ia) and octahydrohyperforin (Ib) defined below:

(Ia：R＝CH₃

Ib：R＝CH₂CH₃)

b) preferred compounds of formula (I) as defined in (1) are those wherein R is₂Compounds that are hydrogen (tetrahydrohyperforin Ic and tetrahydrohyperforin Id defined below), most preferably tetrahydrohyperforin (Ic):

(Ic：R＝CH₃

Id：R＝CH₂CH₃)

c) preferred compounds of formula (I) as defined in (1) are those wherein R is₂Compounds that are hydrogen (dodecahydro-hyperforin Ie and dodecahydro-hyperforin If defined below), most preferably dodecahydro-hyperforin (Ie):

(Ie)：R＝CH₃

(If)：R＝CH₂CH₃

a) further preferred compounds of the formula (I) as defined in (1) are those wherein R is₂A compound of lithium: (Octahydrohyperforin lithium salt Ig and octahydro hyperforin lithium salt Ih), most preferably octahydrohyperforin lithium salt (Ig):

(Ig：R＝CH₃

Ih：R＝CH₂CH₃)

a) further preferred compounds of the formula (I) as defined in (1) are those wherein R is₂Compounds that are acetyl groups (acetooctahydro hyperforin Ii and acetooctahydro hyperforin Il), most preferably acetooctahydro hyperforin (Ii):

(Ii：R＝CH₃

Il：R＝CH₂CH₃)

dodecahydro-hyperforin (Ie), dodecahydro-hyperforin (If), acetooctahydro-hyperforin (Ii) and acetooctahydro-hyperforin (Il) are novel compounds and are also part of the present invention.

The compounds of formulae (Ia) and (Ib) are obtained by catalytic hydrogenation reduction of the isoprene side chain, for example using palladium on charcoal or raney nickel.

Compounds of formulae (Ic) and (Id) are prepared by reacting a compound selected from, for example, NaBH₄、Redal^®、Vitride^®、LiAlH₄Is obtained by reducing the ketone groups at the 1 and 10 positions.

The compounds of formulae (Ie) and (If) are obtained by reducing the isoprene side chain first and then the keto groups in the 1 and 10 positions as described above.

Wherein R is₂Is an inorganic or organic base cation or acyl residueA compound of formula (I) wherein R is₂The compounds of formula (I) which are hydrogen are prepared by conventional methods via salt formation or esterification.

The steps for preparing the compound of the invention by taking hypericum perforatum branches as raw materials are summarized as follows:

hypericum perforatum branches can be extracted by alcohol or aliphatic ketone or mixture of the Hypericum perforatum branches and water or gas under supercritical condition; the resulting extract was partitioned between n-hexane and aqueous fatty alcohol. The hexane solution is extracted with alkaline methanol to extract hyperforin and adhyperforin. The methanolic solution is acidified and then treated with a weakly basic ion exchange resin which selectively retains hyperforin and adhyperforin. The resin was eluted with acidic methanol and the eluate was concentrated to small volume, then diluted with water and back-extracted with n-hexane. The hexane solution was concentrated to a small volume and the resulting concentrate was ready for derivatization. The residue was taken up in a chlorine-containing solvent and then the appropriate reactants were added according to the procedure described in the examples.

The compounds of the invention have shown antidepressant action and are evaluated in rats by a forced swimming test with the evaluation parameters: struggle, float and swim, as described by Cervo et al, Neuropharmacology, 26, 14969-72, 1987. Compounds were administered in 3 divided doses: 30 minutes after the pre-test, 5 hours and 30 minutes before the test. The results, listed in the table below, demonstrate that the compounds of the present invention are more active than the parent compound hyperforin.

Treat mg/kg	Struggle (second)	Float (second)	Swimming (second))
				Carrier	7.0±2.4	174.5±15.9	118.5±15.8
Lithium salt of octahydro hyperforin 6.25	63.1±5.8	59.5±11.3	177.4±14.9
				Tetrahydrocerussin 6.25	51.4±4.1	68.4±7.6	193.4±13.2
Dodecahydro hyperforin 6.25	62.13±5.1	55.1±6.2	169.5±10.1
				Acetyl octahydro hyperforin 6.25	73.9±5.9	68.4±5.7	171.9±11.4
Hyperforin 6.25	30.4±4.6	60.4±7.3	99.3±10.6
				Dixipamine 10	148.3±12.6	53.0±9.2	98.8±7.9

The compounds of the invention also exhibit significant activity against alzheimer's disease due to their ability to increase APPs, a soluble, harmless form of Alzheimer's Precursor Protein (APP). It is in fact known that proteolytic cleavage of Alzheimer Precursor Protein (APP) is regulated both by β -and γ -secretases, which induce an increase in the production of amyloid peptide Ab1-42 (which also has a central role in the characterization of alzheimer's disease), and by α -secretases, which increase the amount of soluble APPs without pathogenic activity (Eslr w.p., Wolfe m.s., Science, 293, 1449-54, 2001).

The effect of the compounds of the invention on the release of APPs by alpha-secretase was evaluated from the neuroblastoma cell line in culture (SH-SY5Y) according to the procedure described in Galbeta J.L.et al, Biochem J.348, 307-313, 2000.

The results, listed in the table below, show that the experimental compounds activate α -secretase mediated APP metabolism, inducing an increase in secreted APPs in the culture medium.

	APPs％
		Control	100
10 mu M hyperforin	296
		10 μ M lithium salt of octahydrohyperforin	1383
10 mu M Tetrahydropipenserin	926
		10 mu M of dodecahydro hyperforin	879
10 mu M acetyloctahydrohyperforin	954

The compounds of the invention may be formulated according to conventional methods, for example as described in Remington's pharmaceutical Sciences Handbook, 17 th edition, Mack pub., n.y., u.s.a, into soft gelatin capsules, hard gelatin capsules, tablets, suppositories; preferably, the extract of the present invention is formulated into a soft gelatin capsule or a controlled release formulation. The dosage range for conventional formulations is 10 to 100mg per unit dose, and controlled release formulations can be as high as 200mg, with a dosage of 200mg per dose per day being suggested here. In addition, the compounds may be administered by a controlled release transdermal route, applying the formulation to the area adjacent to the cerebral carotid artery shunt. The dosage of the compound in these formulations ranges from 10 to 100mg per dose per day.

The present invention will be described in more detail with reference to the following examples.

Examples

EXAMPLE 1 preparation of hyperforin

Extracting 10kg of Hypericum perforatum branches with 30L of methanol in 50L of extraction equipment, and standing the content at room temperature for 3 hours; the extraction was repeated 3 more times, and then the combined extracts were concentrated under vacuum to 5kg and the concentrate was extracted with 3X 5L hexane. The water-methanol solution was discarded, and the hexane solution was back-extracted with basic methanol (KOH) until hyperforin and adhyperforin were absent.

Neutralizing the solution and filtering with weak base ion exchange resin, which can selectively retain hyperforin and adhyperforin; the retained product was again eluted with methanol acidified with phosphoric acid; the methanol eluate was concentrated at 25 ℃ under vacuum, diluted with water and back-extracted with n-hexane until hyperforin was absent.

The combined organic layers were decolorized with 0.3% charcoal and then Na₂SO₄Dried and concentrated in vacuo to an oil at a temperature below 40 ℃. After solidification the oil became a wax (0.52kg) containing about 90% hyperforin.

EXAMPLE 2 preparation of octahydrohyperforin dicyclohexylammonium salt

50g of hyperforin obtained as described in example 1 were dissolved in 500ml of ethyl acetate in the presence of 2g of 5% palladium on charcoal and hydrogenated until complete hydrogen absorption. The catalyst was filtered off, the solution was concentrated to dryness under vacuum and the residue was dissolved in n-hexane. A stoichiometric amount of dicyclohexylamine is added to the solution to selectively crystallize the corresponding salt.

62g of octahydrohyperforin dicyclohexylammonium salt are obtained, the spectral characteristics of which are as follows:

¹H-NMR(300MHz CDCl₃)：δ3.03(2H，m，CH-DCHA)，2.55-2.30，2.10-1.76(20H，m，CH₂-DCHA)，1.70-1.10(22H，m，H-4，H-11，CH₂-5，CH₂-15，CH₂-16，CH₂-17，CH₂-21，CH₂-22，CH₂-26，CH₂-27，CH₂-31，CH₂-32)，0.97-0.83(24H，d，CH₃-19，CH₃-20，CH₃-24，CH₃-25，CH₃-29，CH₃-30，CH₃-34，CH₃-35)，1.19，1.12(6H，d，J＝6.5Hz，CH₃-12，CH₃-13)，0.91(3H，s，CH₃-14)。

¹³C-NMR(75MHz CDCl₃)：δ213.1，211.1，186.3，183.6，119.0，82.5，60.8，53.5，47.5，44.2，41.3，41.0，40.9，38.2，38.1，37.8，33.8，31.0，30.7，30.0，29.4，28.8，28.3，27.9，27.1，25.4，25.1，24.9，23.5，23.2，23.1，22.9，22.8，22.7，22.5，13.7。ESIMS m/z 567[M+Na⁺](100)，1111[2M+Na⁺](91)。

EXAMPLE 3 preparation of Tetrahydropipennin

2g of hyperforin (m.w. ═ 536,01) were dissolved in 20ml of THF under magnetic stirring; adding a large excess of LiAlH to the solution₄(1g, 0.026mol, m.w. ═ 38). The progress of the reaction was monitored by TLC (eluent: petroleum ether/EtOAc 9: 1). The reaction was complete after ten minutes.

Adding Na attached to diatomite₂S₂O₄·10H₂O (3: 1 weight ratio) to destroy excess reactants: the reaction is highly exothermic and therefore requires cooling with ice. Part of the solvent evaporates due to the heat generated. Mixing the raw materialsThe material was filtered through celite and the filtrate was washed three times with 20ml AcOEt. The solution was placed in a 150ml round-bottomed, necked flask and the solvent was evaporated off completely.

The resulting mixture was purified by column chromatography using 200ml column packed with 100ml silica gel and petroleum ether/EtOAc 95: 5 as the elution mixture. Fractions of about 20ml of eluate were collected and their composition was checked by TLC (petroleum ether/EtOAc 9: 1). The spectral properties of the higher content product crystallized from methanol are as follows:

¹H-NMR(300MHz CDCl₃)：δ5.11(1H，m，H-22)，5.00(3H，m，H-17，H-27，H-32)，3.11(1H，dd，J＝14.0，7.4Hz，CH₂-26)，2.92(1H，dd，J＝14.0，7.0Hz，CH₂-26)，2.50-1.35(12H，m，H-4，H-11，CH₂-5，CH₂-15，CH₂-16，CH₂-21，CH₂-31)，1.80-1.52(24H，s，CH₃-19，CH₃-20，CH₃-24，CH₃-25，CH₃-29，CH₃-30，CH₃-34，CH₃-35)，1.19-0.95(9H，d，CH₃-12，CH₃-13，CH₃-14)。

¹³C-NMR(75MHz CDCl₃)：δ200.5，174.3，134.1，132.6，131.2 130.6，125.8，123.9，122.6，120.5，119.4，79.2，73.1，39.6，37.2，30.5，32.8，31.3，30.2，26.1，26.0，25.8，23.5，23.1，21.9，20.0，18.3，18.1，17.8，15.6。ESIMS m/z 1103[2M+Na⁺](100)，541[M+H⁺](25)，563[M+Na⁺](12)。

EXAMPLE 4 preparation of lithium salt of octahydro-hyperforin

15g of octahydrohyperforin dicyclohexylammonium salt were eluted on acidic resin (Dowex50X8, 300g) with 600ml of methanol. 11.01g of octahydrohyperforin was obtained, to which 0.8745g of LiOH monohydrate dissolved in water was added. The mixture was evaporated to dryness to give 11.41g of lithium salt, whose spectral characteristics were as follows:

¹H-NMR(300MHz CDCl₃)：δ1.93-1.00(22H，m，H-4，H-11，CH₂-5，CH₂-15，CH₂-16，CH₂-17，CH₂-21，CH₂-22，CH₂-26，CH₂-27，CH₂-31，CH₂-32)，1.00-0.80(24H，d，CH₃-19，CH₃-20，CH₃-24，CH₃-25，CH₃-29，CH₃-30，CH₃-34，CH₃-35)，1.20，1.06(6H，d，J＝6.3Hz，CH₃-12，CH₃-13)，0.91(3H，s，CH₃-14)。

¹³C-NMR(75MHz CDCl₃)：δ211.4，191.3，184.6，82.7，61.5，51.3，47.7，41.5，40.5，38.2，37.9，37.7，33.9，30.5，29.6，28.7，28.3，28.1，27.1，23.3，23.1，23.0，22.8，22.7，22.4，22.0，14.0。ESIMS m/z 551[M+H⁺](100)，557[M+Li⁺](40)，1102[2M+H⁺](71)，1108[M+Li⁺](75)。

EXAMPLE 5 preparation of dodecahydro-hyperforin

1.72g of octahydrohyperforin dicyclohexylammonium salt (m.w. ═ 716; 2.41mmol) are dissolved in 20ml of THF under magnetic stirring; a large excess (3.5g) of LiAlH was added to the solution₄(m.w. ═ 38; 0.092 mol). The progress of the reaction was monitored by TLC (eluent: petroleum ether/EtOAc 9: 1). The reaction was complete after ten minutes.

Excess reactants were destroyed as described in example 5. The mixture was filtered and the residue was washed thoroughly with ethyl acetate. The solution was evaporated to dryness, the crude reaction product was dissolved in 15ml of petroleum ether/diethyl ether 3: 1 and the solution was placed in a 150ml separatory funnel. The organic phase was washed three times with 2N sulfuric acid and then with brine. Discarding the aqueous phase and the organic phase with Na₂SO₄Dried and concentrated to dryness. The product obtained is purified by column chromatography on 75g of silica gel, eluting the desired compound with petroleum ether/ethyl acetate 99: 1. 0.9g of dodecahydro-hyperforin is obtained, the spectral characteristics of which are as follows:EIMS m/z 548[M]⁺。

EXAMPLE 6 preparation of Acetyloctahydrohyperforin

300mg of acetylhyperforin (m.w. ═ 578; 0.52mmol) was dissolved in 3ml of MeOH in a two-necked round bottom flask, and then the catalyst (5% palladium on charcoal) was added. The reaction was monitored by TLC (petroleum ether/EtOAc 95: 5 Rfp ═ 0.43; Rfa ═ 0.52). The reaction was complete after four hours. The catalyst was filtered off through a layer of celite and the methanol was then distilled off.

The reaction product is purified by column chromatography on 30g of silica gel, eluting with a petroleum ether/ethyl acetate 9: 1 mixture. Crystallization from methanol gave 150mg of the desired compound, whose spectral characteristics were as follows: EIMS M/z 586[ M ]]⁺。

Claims (12)

1. Hyperforin and adhyperforin derivatives of formula (I) for use as a medicament,

wherein R is methyl or ethyl, and wherein:

i)R₂is a pharmaceutically acceptable inorganic or organic base cation or a straight chain or branched chain C₂-C₅Acyl residue, R₁Is 3-methyl-but-1-yl and oxo is present in the 1 and 10 positionsClustering; or

ii)R₂Is hydrogen, a pharmaceutically acceptable cation of an inorganic or organic base or a straight or branched chain C₂-C₅Acyl residue, R₁Is 3-methyl-but-1-yl and hydroxyl groups are present in the 1 and 10 positions.

2. The derivative of claim 1, wherein R₂Is lithium, R₁Is 3-methyl-but-1-yl and oxo groups are present in the 1-and 10-positions.

3. The derivative of claim 1, wherein R₂Is acetyl, R₁Is 3-methyl-but-1-yl and oxo groups are present in the 1-and 10-positions.

4. The derivative of claim 2 or 3, wherein R is methyl.

5. The derivative of claim 1, wherein R₂Is hydrogen, R₁Is 3-methyl-but-1-yl and hydroxyl groups are present in the 1 and 10 positions.

6. A compound is selected from the group consisting of dodecahydro hyperforin (Ie), dodecahydro hyperforin (If), octahydro hyperforin lithium salt (Ig) and octahydro hyperforin (Ih), acetyloctahydro hyperforin (Ii) and acetyloctahydro hyperforin (Il).

7. Pharmaceutical compositions comprising hyperforin and adhyperforin derivatives of formula (I),

wherein R is methyl or ethyl, R₂Is hydrogen, a pharmaceutically acceptable cation of an inorganic or organic base or a straight or branched chain C₂-C₅An acyl residue, and wherein:

a)R₁is a 3-first-but-1-yl and oxo groups are present in positions 1 and 10; or

b)R₁Is 3-methyl-2-buten-1-yl and hydroxyl groups are present at the 1 and 10 positions; or

c)R₁Is 3-methyl-but-1-yl and hydroxyl groups are present in the 1 and 10 positions.

8. The composition of claim 7, wherein R₂Is hydrogen, R₁Is 3-methyl-but-1-yl and oxo groups are present in the 1-and 10-positions.

9. The composition of claim 7, wherein R₂Is hydrogen, R₁Is 3-methyl-2-buten-1-yl, and hydroxyl groups are present at the 1-and 10-positions.

10. The composition of claim 8 or 9, wherein R is methyl.

11. The use of hyperforin and adhyperforin derivatives of formula (I) for the preparation of a medicament for the treatment of depression and Alzheimer's disease,

wherein R is methyl or ethyl, R₂Is hydrogen, a pharmaceutically acceptable cation of an inorganic or organic base or a straight or branched chain C₂-C₅An acyl residue, and wherein:

a)R₁is 3-methyl-but-1-yl and oxo groups are present in positions 1 and 10; or

b)R₁Is 3-methyl-2-buten-1-yl and hydroxyl groups are present at the 1 and 10 positions; or

c)R₁Is 3-methyl-but-1-yl and hydroxyl groups are present in the 1 and 10 positions.

12. The use of claim 11, wherein said derivative is selected from the group consisting of octahydrohyperforin (Ia), octahydrohyperforin (Ib), tetrahydrohyperforin (Ic), tetrahydrohyperforin (Id), dodecahydro-hyperforin (Ie), dodecahydro-hyperforin (If), octahydrohyperforin lithium salt (Ig), octahydrohyperforin lithium salt (Ih), acetyloctahydrohyperforin (Ii), and acetyloctahydrohyperforin (Il).